Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02340167 2001-02-08
1
NEW CARBOXYLIC ACID DERIVATIVES CARRYING KETO
SIDE-CHAINS, THEIR PRODUCTION AND THEIR USE
AS ENDOTHELIN-RECEPTOR ANTAGONISTS
The present invention relates to novel carboxylic acid
derivatives, their preparation and use.
Endothelia is a peptide constructed from 21 amino acids, which is
synthesized and released by vascular endothelium. Endothelia '
exists in three isoforms, ET-1, ET-2 and ET-3. Below,
"endothelia" or "ET" designates one or all isoforms of
endothelia. Endothelia is a potent vasoconstrictor and has a
strong effect on vascular tone. It is known that this
vasoconstriction is caused by the binding of endothelia to its
receptor (Nature, 332, 411-415, 1988; FEBS Letters, ~, 440-444,
1988 and Biochem. Biophys. Res. Commun., 154, 868-875, 1988).
Increased or abnormal release of endothelia causes a lasting
vascular contraction in peripheral, renal and cerebral blood
vessels, which can lead to diseases. As reported in the
literature, endothelia is involved in a number of illnesses.
These include: hypertension, acute myocardial infarct, pulmonary
hypertension, Raynaud's syndrome, cerebral vasospasms, stroke,
benign prostate hypertrophy, atherosclerosis and asthma (J.
Vascular Med. Biology 2_, 207 (1990), J. Am. Med. Association 264,
2868 (1990), Nature 344, 114 (1990), N. Engl. J. Med. 322, 205
(1989), N. Engl. J. Med. 328, 1732 (1993), Nephron 66, 373
(1994), Stroke 25, 904 (1994), Nature 365, 759 (1993), J. Mol.
Cell. Cardiol. 27, A234 (1995); Cancer Research 56, 663 (1996)).
At least two endothelia receptor subtypes, the ETA and ETB
receptor, have been described in the literature at present
(Nature 348, 730 (1990), Nature 348, 732 (1990)). Accordingly,
substances which inhibit the binding of endothelia to both
receptors should antagonize physiological effects of endothelia
and therefore be valuable pharmaceuticals.
Mixed endothelia receptor antagonists are those compounds which
bind to the ETA and ETB receptor with approximately equal
affinity. Approximately equal affinity for the receptors exists
when the quotient of the affinities is greater than 0.05
CA 02340167 2001-02-08
la
(preferably 0.1) and smaller than 20 (preferably 10).
The patent application DE 19636046.3 describes mixed ETA/ET$
receptor antagonists. The spacer Q (see formula XX), which
corresponds in length to a Cz-C4-alkyl chain, is important for
these compounds.
CA 02340167 2001-02-08
0050/49265
2
R2
R4
I H
R6-Q-W-C-C-O-~~ Z XX
RS Rl X =
R3
Mixed receptor antagonists are also obtained with the spacer
Q = COCR7R8 (see formula I).
15
The object of the invention is to identify compounds which bind
with approximately equal affinity to the ETA and the ETB receptor
and have more advantageous properties compared with the mixed
endothelin receptor antagonists already known.
The invention relates to carboxylic acid derivatives of the
formula I
O RB R4 R2
II I I g
R6 C-C-W-C-C-O-~~ Z I
R~ RS R1 X ~ 3
R
in which the substituents have the following meanings:
R1 is tetrazole [sic] or a group
O
C-R
R is
a) a radical OR9, in which R9 is:
hydrogen, the cation of an alkali metal, the cation of an
alkaline earth metal, a physiologically tolerable organic
ammonium ion such as tertiary C1-C4-alkylammonium or the
ammonium ion;
C3-C$-cycloalkyl, C1-CB-alkyl, CH2-phenyl, which can be
substituted by one or more of the following radicals:
halogen, nitro, cyano, C1-C4-alkyl, C1-C4-haloalkyl, hydroxyl,
C1-C4-alkoxy, mercapto, C1-C4-alkylthio, amino,
NH(C1-C4-alkyl), N(C1-C4-alkyl)2;
CA 02340167 2001-02-08
0050/49265
3
a C3-C6-alkenyl or a C3-C6-alkynyl group, where these groups
for their part can carry one to five halogen atoms;
a phenyl radical which can carry one to five halogen atoms
and/or one to three of the following radicals: nitro, cyano,
C1-C4-alkyl, C1-C4-haloalkyl, hydroxyl, C1-Cq-alkoxy,
mercapto, C1-C4-alkylthio, amino, NH(C1-C4-alkyl),
N(C1-C4-alkyl)2;
b) a 5-membered heteroaromatic which is linked via a nitrogen
atom, such as pyrrolyl, pyrazolyl, imidazolyl and triazolyl,
and which can carry one or two halogen atoms, or one or two
C1-C4-alkyl [lacuna] or one or two C1-C4-alkoxy groups,
c) a group
k
-O- CH ( SI ) Rlo
2~p
in which k [lacuna] assume the values 0, 1 and [sic] 2, p
(lacuna] assume the values 1, 2, 3 and [sic) 4 and Rlo is
C1-C4-alkyl, C3-C8-cycloalkyl, C3-C6-alkenyl, C3-C6-alkynyl or
phenyl, which can be substituted by one or more, e.g. one to
three, of the following radicals:
halogen, vitro, cyano, C1-C4-alkyl, C1-C4-haloalkyl, hydroxyl,
C1-C4-alkoxy, C1-C4-alkylthio, mercapto, amino,
NH(C1-C4-alkyl), N(C1-C4-alkyl)Z;
d) a radical
O
3 5 -N-S-R1l
H II
O
in which R11 is:
C1-C4-alkyl, C1-C4-haloalkyl, C3-C6-alkenyl, C3-C6-alkynyl,
C3-C$-cycloalkyl, where these radicals can carry a
C1-C4-alkoxy, C1-C4-alkylthio and/or a phenyl radical as
mentioned under c);
CA 02340167 2001-02-08
0050/49265
4
phenyl, which can be substituted by one to three of the
following radicals: halogen, vitro, cyano, C1-C4-alkyl,
C1-C4-haloalkyl, hydroxyl, C1-C4-alkoxy, C1-C4-alkylthio,
mercapto, amino, NH(C1-C4-alkyl), N(C1-C4-alkyl)2;
RZ is hydrogen, hydroxyl, NH2, NH(C1-C4-alkyl), N(C1-C4-alkyl)2,
halogen, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl,
C1-C4-hydroxyalkyl, C1-C4-haloalkyl, C1-C4-alkoxy,
C1-C4-haloalkoxy or C1-C4-alkylthio, or CR2 is linked to CR12 as
indicated under Z to give a 5- or 6-membered ring;
X is nitrogen or methine;
Y is nitrogen or methine;
Z is nitrogen or CR12, in which R12 is hydrogen, halogen,
C1-C4-haloalkyl or C1-Cq-alkyl, or CRlz, together with CRz or
CR3, forms a 5- or 6-membered alkylene or alkenylene ring,
which can be substituted by one or two C1-C4-alkyl groups and
in which one or more methylene groups in each case can be
replaced by oxygen, sulfur, -NH or N(C1-C4-alkyl), where at
least one of the ring members X, Y or Z is nitrogen;
R3 is hydrogen, hydroxyl, NH2, NH(C1-C4-alkyl), N(C1-C4-alkyl)z,
halogen, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl,
C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy,
C1-C4-hydroxyalkyl, C1-C4-alkylthio, or CR3 is linked to CR12 as
indicated under Z to give a 5- or 6-membered ring;
R4 and R5 (which can be identical or different) are:
phenyl or naphthyl, each of which can be substituted by one
or more of the following radicals: halogen, vitro, cyano,
hydroxyl, mercapto, C1-C4-alkyl, C2-C4-alkenyl, CZ-Cq-alkynyl,
C1-C4-haloalkyl, C1-C4-alkoxy, phenoxy, carboxyl,
C1-C4-haloalkoxy, C1-C4-alkylthio, amino, NH(C1-C4-alkyl),
N(C1-C4-alkyl)2 or phenyl, which can be mono- or
polysubstituted, e.g. mono- to trisubstituted, by halogen,
vitro, cyano, C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy,
C1-C4-haloalkoxy or C1-C4-alkylthio;
phenyl or naphthyl, which are connected to one another in the
ortho-position via a direct bond, a methylene, ethylene or
ethenylene group, an oxygen or sulfur atom or an SO2, NH or
N-alkyl group;
CA 02340167 2001-02-08
0050/49265
C3-Cg-cycloalkyl;
R6 is C3-Ce-cycloalkyl, where these radicals in each case can be
mono- or polysubstituted by: halogen, hydroxyl, mercapto,
5 carboxyl, vitro, cyano, C1-Cq-alkoxy, C1-Cq-alkyl,
CZ-Cq-alkenyl, C2-Cq-alkynyl, C3-C6-alkenyloxy,
C3-C6-alkynyloxy, C1-Cq-alkylthio, C1-Cq-haloalkoxy,
C1-Cq-alkylcarbonyl, C1-Cq-alkoxycarbonyl,
C3-C8-alkylcarbonylalkyl, carboxamide, NH(C1-Cq-alkyl),
N(C1-Cq-alkyl)y, or phenyl which can be mono- or
polysubstituted, e.g. mono- to trisubstituted, by halogen,
vitro, cyano, C1-Cq-alkyl, C1-Cq-haloalkyl, C1-Cq-alkoxy,
C1-Cq-haloalkoxy or C1-Cq-alkylthio;
phenyl or naphthyl, each of which can be substituted by one
or more of the following radicals: halogen, R15, vitro,
mercapto, carboxyl, cyano, hydroxyl, amino, C1-Cq-alkyl,
CZ-Cq-alkenyl, C2-Cq-alkynyl, C3-C6-alkenyloxy,
C1-Cq-haloalkyl, C3-C6-alkynyloxy, C1-Cq-alkylcarbonyl,
C1-Cq-alkoxycarbonyl, carboxamide, C1-Cq-alkoxy,
C1-Cq-haloalkoxy, phenoxy, C1-Cq-alkylthio, NH(C1-Cq-alkyl),
N(C1-Cq-alkyl)2, dioxomethylene, dioxoethylene or phenyl,
which can be mono- or polysubstituted, e.g. mono- to
trisubstituted, by halogen, vitro, cyano, C1-Cq-alkyl,
C1-Cq-haloalkyl, C1-Cq-alkoxy, C1-Cq-haloalkoxy or
C1-Cq-alkylthio;
a five- or six-membered heteroaromatic, comprising one to
three nitrogen atoms and/or a sulfur or oxygen atom, which
can carry one to four halogen atoms and/or one or two of the
following radicals: C1-Cq-alkyl, C2-Cq-alkenyl,
C1-Cq-haloalkyl, C1-Cq-alkoxy, C1-Cq-haloalkoxy,
C1-Cq-alkylthio, phenyl or phenoxy, where the phenyl radicals
for their part can carry one to five halogen atoms and/or one
to three of the following radicals: C1-Cq-alkyl,
C1-Cq-haloalkyl, C1-Cq-alkoxy, C1-Cq-haloalkoxy and/or
C1-Cq-alkylthio;
R7 and R$ (which can be identical or different) are:
hydrogen, C1-C4-alkyl;
R15 is C1-C4-alkyl, C1-C4-alkylthio, C1-C4-alkoxy, each of which
carries one of the following radicals: hydroxyl, carboxyl,
amino, NH(C1-C4-alkyl), N(C1-C4-alkyl)2, carboxamide or
CON(C1-C4-alkyl)2;
CA 02340167 2001-02-08
0050/49265
6
W is sulfur or oxygen.
Here and in the following text, the following definitions apply:
An alkali metal is, for example, lithium, sodium or potassium;
An alkaline earth metal is, for example, calcium, magnesium or
barium;
C3-CB-cycloalkyl is, for example, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl;
C1-C4-haloalkyl can be linear or branched such as, for example,
fluoromethyi, difluoromethyl, trifluoromethyl,
chlorodifluoromethyl, dichlorofluoromethyl, trichloromethyl,
1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl,
2,2,2-trifluoroethyl, 2-chloro-2,2-difluoroethyl,
2,2-dichloro-2-fluoroethyl, 2,2,2-trichloroethyl or
pentafluoroethyl;
C1-C4-haloalkoxy can be linear or branched such as, for example,
difluoromethoxy, trifluoromethoxy, chlorodifluoromethoxy,
1-fluoroethoxy, 2,2-difluoroethoxy, 1,1,2,2-tetrafluoroethoxy,
2,2,2-trifluoroethoxy, 2-chloro-1,1,2-trifluorethoxy,
2-fluoroethoxy or pentafluoroethoxy;
C1-C4-alkyl can be linear or branched such as, for example,
methyl, ethyl, 1-propyl, 2-propyl, 2-methyl-2-propyl,
2-methyl-1-propyl, 1-butyl or 2-butyl;
C2-C4-alkenyl can be linear or branched such as, for example,
ethenyl, 1-propen-3-y1, 1-propen-2-yl, 1-propen-1-yl,
2 methyl-1-propenyl, 1-butenyl or 2-butenyl;
Cz-C4-alkynyl can be linear or branched such as, for example,
ethynyl, 1-propyn-1-yl, 1-propyn-3-yl, 1-butyn-4-yl or
2-butyn-4-yl;
C1-C4-alkoxy can be linear or branched such as, for example,
methoxy, ethoxy, propoxy, l.-methylethoxy, butoxy,
1-methylpropoxy, 2 methylpropoxy or 1,1-dimethylethoxy;
C3-C6-alkenyloxy can be linear or branched such as, for example,
allyloxy, 2-buten-1-yloxy or 3-buten-2-yloxy;
CA 02340167 2001-02-08
0050/49265
7
C3-C6-alkynyloxy can be linear or branched such as, for example,
2-propyn-1-yloxy, 2-butyn-1-yloxy or 3-butyn-2-yloxy;
C1-C4-alkylthio can be linear or branched such as, for example,
methylthio, ethylthio, propylthio, 1-methylethylthio, butylthio,
1-methylpropylthio, 2-methylpropylthio or 1,1-dimethylethylthio;
C1-C4-alkylcarbonyl can be linear or branched such as, for
example, acetyl, ethylcarbonyl or 2-propylcarbonyl;
C1-C4-alkoxycarbonyl can be linear or branched such as, for
example, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl,
i-propoxycarbonyl or n-butoxycarbonyl;
C3-Ce-alkylcarbonylalkyl can be linear or branched, e.g.
2-oxoprop-1-yl, 3-oxobut-1-yl or 3-oxobut-2-yl;
C1-C$-alkyl can be linear or branched such as, for example,
C1-C4-alkyl, pentyl, hexyl, heptyl or octyl;
halogen is, for example, fluorine, chlorine, bromine or iodine.
The invention further relates to those compounds from which the
compounds of the formula I can be released (so-called prodrugs).
30
Preferred prodrugs are those in which the release takes place
under those conditions which prevail in certain body
compartments, e.g. in the stomach, intestine, blood circulation
or liver.
The compounds I and also the intermediates for their preparation,
such as, for example, II, III, IV, V and VI, can have one or more
asymmetrically substituted carbon atoms. Such compounds can be
present as pure enantiomers or pure diastereomers or as a mixture
thereof. The use of an enantiomerically pure compound as an
active compound is preferred.
The invention further relates to the use of the abovementioned
carboxylic acid derivatives for the production of drugs, in
particular for the production of inhibitors for ETA and ETB
receptors. The compounds according to the invention are suitable
as mixed antagonists, as were defined at the outset.
The preparation of the compounds having the general formula IV in
which W is sulfur or oxygen can be carried out as described in WO
96/11914.
CA 02340167 2001-02-08
0050/49265
8
In this reaction, the later keto group is protected as a cyclic
acetal; however, other protective groups are also conceivable,
such as, for example, dimethyl acetal.
O O O R8 O O RB R4
R4~%~ Rl \ / I \ / I I H
L~ .~- C-C-W-H ---~ C-C-W-C-C-OH
Rs Rs~ R~ Rs/ R~ Rs Ri
II III IV
Compounds of the formula IV can be obtained in enantiomerically
pure form by starting from enantiomerically pure compounds of the
formula II and reacting them with compounds of the formula III as
described in WO 96/11914.
Furthermore, enantiomeric compounds of the formula IV can be
obtained by carrying out a classical resolution with suitable
enantiomerically pure bases using racemic or diastereomeric
compounds of the formula IV. Suitable bases of this type are, for
example, 4-chlorophenylethylamine and bases such as are mentioned
in WO 96/11914.
The preparation of compounds of the general formula II was
described in WO 96/11914, while compounds of the general formula
III are either known or can be synthesized by generally known
methods such as, for example:
O s
O R 1. ) R6MgSr \ /
Me0\ ~C-C-W C-C-W-H
I~ 2.) Deprotection si I
7
R 3.) Acetalization R R III
Carboxylic acid derivatives of the general formula IV can then be
reacted with compounds of the general formula V, substances of
the type VI being obtained.
RZ ~ 0 RB R4 Y ~ 2
Y
IV -f- R16 ~ ~Z --~- C-C-W-C-C-O--~~ \Z
R6~ R7 Rs Rl X ~ 3
X-~
'R3 R
V VI
CA 02340167 2001-02-08
0050/49265
9
In formula V, R16 is halogen or R1~-S02-, where R1~ can be
C1-C4-alkyl, C1-C4-haloalkyl or phenyl. Furthermore, at least one
of the ring members X or Y or Z is nitrogen. The reaction
preferably takes place in an inert solvent or diluent with
addition of a suitable base, i.e. of a base which brings about a
deprotonation of the intermediate IV, in a temperature range from
room temperature up to the boiling point of the solvent.
Compounds of the type VI with R1 = COOH can be obtained directly
in this manner if the intermediate IV, in which R1 is COON, is
deprotonated using two equivalents of a suitable base and reacted
with compounds of the general formula V. Here too, the reaction
takes place in an inert solvent and in a temperature range from
room temperature up to the boiling point of the solvent.
Examples of such solvents or diluents are aliphatic, alicyclic
and aromatic hydrocarbons, each of which can be optionally
chlorinated, such as, for example, hexane, cyclohexane, petroleum
ether, naphtha, benzene, toluene, xylene, methylene chloride,
chloroform, carbon tetrachloride, ethyl chloride and
trichloroethylene, ethers, such as, for example, diisopropyl
ether, dibutyl ether, methyl tert-butyl ether, propylene oxide,
dioxane and tetrahydrofuran, nitriles, such as, for example,
acetonitrile and propionitrile, acid amides, such as, for
example, dimethylformamide, dimethylacetamide and
N-methylpyrrolidone, and sulfoxides and sulfones, such as, for
example, dimethyl sulfoxide and sulfolane.
Compounds of the formula V are known, in some cases commercially
available or can be prepared in a generally known manner.
The base used can be an alkali metal or alkaline earth metal
hydride such as sodium hydride, potassium hydride or calcium
hydride, a carbonate such as an alkali metal carbonate, e.g.
sodium or potassium carbonate, an alkali metal or alkaline earth
metal hydroxide such as sodium or potassium hydroxide, an
organometallic compound such as butyllithium or an alkali metal
amide such as lithium diisopropylamide or lithium amide.
The compounds according to the invention, in which the
substituents have the meanings indicated under the general
formula I, can finally be prepared by removing the keto
protective group in the compounds of the formula VI. In the case
of the ethylene glycol acetal, this can be carried out by acidic
hydrolysis.
CA 02340167 2001-02-08
0050/49265
O R$ R4 R2
II I I H
VI -~ R6 C-C-W-C-C-O-~~ Z
5 R~ Rs Rl X ~ s
R
I
Compounds of the type I can furthermore be synthesized via
10 compounds having the formula VII
O R8
II I
1. ) Me0 C - C - W -H RZ
Iz O R8 R4 ~J
I R I ) I I H Y \\
II Me0\ ~C-C-W-C-C-O-~~ Z
2 . ) V i R' RS Rl x Rs
VII
The compounds having the general formula VII can then be reacted
with Grignard reagents to give the compounds of the formula I
Rx
O R8 R4
II I I H
VII R-MgBr R6 C--C-W-C-C-O-~~ Z
R' R5 R1 X ~ s
I R
Compounds of the formula I can also be prepared by starting from
the corresponding carboxylic acids, i.e. compounds of the formula
I in which R1 is COON, and first converting these in a customary
manner into an activated form such as an acid halide, an
anhydride or imidazolide and then reacting this with an
appropriate hydroxyl compound HORS. This reaction can be carried
out in the customary solvents and often necessitates the addition
of a base, those mentioned above being suitable. These two steps
can also be simplified, for example, by allowing the carboxylic
acid to act on the hydroxyl compound in the presence of a
dehydrating agent such as a carbodiimide.
Moreover, compounds of the formula I can also be prepared by
starting from the salts of the corresponding carboxylic acids,
i.e. from compounds of the formula I in which R1 is a group COOM,
where M can be an alkali metal cation or the equivalent of an
alkaline earth metal cation. These salts can be reacted with many
compounds of the formula R9-A, A being a customary nucleofugic
CA 02340167 2001-02-08
0050/49265
20
30
11
leaving group, for example halogen such as chlorine, bromine,
iodine or aryl- or alkylsulfonyl optionally substituted by
halogen, alkyl or haloalkyl, such as, for example,
toluenesulfonyl and methylsulfonyl or another equivalent leaving
5 group. Compounds of the formula R9-A having a reactive substituent
A are known or easy to obtain using the general expert knowledge.
This reaction can be carried out in the customary solvents and is
advantageously performed with addition of a base, those mentioned
above being suitable.
In some cases, the use of generally known protective group
techniques is necessary for the preparation of the compounds I
according to the invention. If, for example, R6 is to be
4-hydroxyphenyl, the hydroxyl group can first be protected as a
15 benzyl ether, which is then cleaved at a suitable stage in the
reaction sequence.
Compounds of the formula I in which R1 is tetrazole [sic] can be
prepared as described in WO 96/11914.
With respect to the biological action, carboxylic acid
derivatives of the general formula I - both as pure enantiomers
or pure diastereomers or as a mixture thereof - are preferred in
which the substituents have the following meanings:
R2 is hydrogen, N(C1-C4-alkyl)2, C1-C4-alkyl, C1-C4-alkoxy,
C1-C4-alkylthio, C1-C4-haloalkyl, C1-C4-haloalkoxy,
hydroxymethyl or CR2 is linked to CR12 as indicated under Z to
give a 5- or 6-membered ring;
X is nitrogen or methine;
Y is nitrogen or methine;
Z is nitrogen or CR12, in which R12 is hydrogen, fluorine,
trifluoromethyl or methyl or CRlz, together with CR2 or CR3,
forms a 5- or 6-membered alkylene or alkenylene ring which
can be substituted by one or two methyl groups, and in which
one methylene group in each case can be replaced by oxygen or
sulfur, such as --CH2-CHZ-O-, -CHZ-CH2-CH2-O-, --CH=CH-0-,
--CH=CH-CHzO-, --CH ( CH3 )--CH ( CH3 )-Q-, --CH=C ( CH3 )--0-,
-C{CH3)=C(CH3)-O- or -C(CH3)=C(CH3)-S;
at least one of the ring members X, Y or Z is nitrogen;
CA 02340167 2001-02-08
0050/49265
12
R3 is hydrogen, N(C1-C4-alkyl)2, C1-C4-alkyl, C1-C4-alkoxy,
C1-CQ-alkylthio, C1-C4-haloalkyl, CI-C4-haloalkoxy,
hydroxymethyl or CR3 is linked to CR12 linked as indicated
under Z to give a 5- or 6-membered ring;
R4 and R5 (which can be identical or different) are:
phenyl or naphthyl, each of which can be mono- to
trisubstituted by halogen, cyano, C1-C4-alkyl,
C1-C4-haloalkyl, C1-C4-alkoxy, phenoxy, C1-C4-alkylthio,
NH(C1-C4-alkyl) or N(C1-C4-alkyl)2 or phenyl, which can be
mono- to trisubstituted by halogen, cyano, C1-C4-alkyl,
C1-C4-haloalkyl, C1-C4-alkoxy, C1-C4-haloalkoxy or
C1-C4-alkylthio; or
phenyl or naphthyl, each of which can be bonded to one
another in the ortho-position via a direct bond, a methylene,
ethylene or ethenylene group, an oxygen or sulfur atom or an
S02-, NH- or N-alkyl-group;
C5-C6-cycloalkyl;
R6 C3-Cg-cycloalkyl, where these radicals [sic] in each case can
be mono- to trisubstituted by: halogen, C1-C4-alkoxy,
C1-C4-alkyl, C1-C4-alkylthio, C1-C4-haloalkoxy,
C1-C4-alkoxycarbonyl or phenyl, which can be mono- to
trisubstituted by halogen, C1-C4-alkyl, C1-C4-haloalkyl,
C1-C4-alkoxy, C1-C4-haloalkoxy or C1-C4-alkylthio;
phenyl or naphthyl, each of which can be mono- to
trisubstituted by halogen, R15, cyano, hydroxyl, C1-C4-alkyl,
C1-C4-haloalkyl, C1-C4-alkylcarbonyl, C1-C4-alkoxycarbonyl,
C1-C4-alkoxy, C1-C4-haloalkoxy, phenoxy, C1-C4-alkylthio,
NH(C1-C4-alkyl), N(CZ-C4-alkyl)2, dioxomethylene,
dioxoethylene or phenyl, which can be mono- to trisubstituted
by halogen, cyano, C1-C4-alkyl, C1-C4-haloalkyl, C1-C4-alkoxy,
C1-C4-haloalkoxy or C1-C4-alkylthio;
a five- or six-membered heteroaromatic, comprising one to
three nitrogen atoms and/or a sulfur or oxygen atom which can
carry one or two halogen atoms and/or one or two of the
following radicals: C1-C4-alkyl, C1-C4-alkoxy,
trifluoromethoxy, C1-C4-alkylthio, phenyl or phenoxy, where
the phenyl radicals for their part can carry one to five
halogen atoms and/or one to three of the following radicals:
C1-C4-alkyl, C1-C4-alkoxy and/or C1-C4-alkylthio;
CA 02340167 2001-02-08
0050/49265
13
R7 and RB (which can be identical or different) are:
hydrogen, C1-C4-alkyl;
R15 is methyl, ethyl, methoxy or ethoxy, each of which carries
one of the following radicals: hydroxyl, carboxyl, amino,
NH(C1-C4-alkyl), N(C1-C4-alkyl)2, carboxamide or
CON(C1-C4-alkyl)2;
W is sulfur or oxygen.
Particularly preferred compounds of the formula I - both as pure
enantiomers or pure diastereomers or as a mixture thereof - are
those in which the substituents have the following meanings:
R2 is trifluoromethyl, C1-C4-alkyl, C1-C4-alkoxy,
C1-C4-alkylthio, or CR2 is linked to CR12 as indicated under Z
to give a 5- or 6-membered ring;
X is nitrogen or methine;
Y is nitrogen or methine;
Z is nitrogen or CR12, in which R1z is hydrogen, fluorine or
methyl or CR12, together with CR2 or CR3, forms a 5- or
6-membered alkylene or alkenylene ring, in which one
methylene group in each case can be replaced by oxygen or
sulfur, such as -CH2-CHZ-S-, --CH=CH-O-, -CHZ-CH2-S- [sic];
at least one of the ring members X, Y or Z is nitrogen;
R3 is trifluoromethyl, C1-C4-alkyl, C1-C4-alkoxy, C1-C4-alkylthio
or CR3 is linked to CR12 as indicated under Z to give a 5- or
6-membered ring;
R4 and R5 (which can be identical or different) are:
phenyl or naphthyl, each of which can be mono- to
trisubstituted by halogen, C1-C4-alkyl, C1-C4-alkoxy, phenoxy
or phenyl, which can be mono- to trisubstituted by halogen,
C1-C4-alkyl or C1-C4-alkoxy; or
phenyl or naphthyl, which can be bonded to one another in the
ortho-position by a direct bond, a methylene, ethylene or
ethenylene group or an SOZ group;
CA 02340167 2001-02-08
0050/49265
14
cyclohexyl;
R6 is cyclohexyl which can be mono- to trisubstituted by
C1-C4-alkoxy, C1-C4-alkyl, halogen or phenyl, which can be
mono- to trisubstituted by halogen, C1-C4-alkyl, C1-C4-alkoxy;
phenyl or naphthyl, which in each case can be mono- to
trisubstituted by halogen, R15, C1-C4-alkyl, C1-C4-haloalkyl,
acetyl, C1-C4-alkoxycarbonyl, C1-C4-alkoxy, phenoxy,
C1-C4-alkylthio, dioxomethylene, dioxoethylene or phenyl,
which can be mono- to trisubstituted by halogen, C1-C4-alkyl,
C1-C4-alkoxy, or C1-C4-alkylthio;
R~ and Rs (which can be identical or different) are:
hydrogen, C1-C4-alkyl;
R15 is methoxy or ethoxy, each of which carries one of the
following radicals: hydroxyl, carboxyl, carboxamide or
CON(C1-C4-alkyl)2;
W is sulfur or oxygen.
The compounds of the present invention offer a novel therapeutic
potential for the treatment of hypertension, pulmonary high blood
pressure, myocardial infarct, angina pectoris, arrhythmia,
acute/chronic kidney failure, chronic cardiac insufficiency,
renal insufficiency, cerebral vasospasms, cerebral ischemia,
subarachnoid hemorrhages, migraine, asthma, atherosclerosis,
endotoxic shock, endotoxin-induced organ failure, intravascular
coagulation, restenosis after angioplasty and bypass operations,
benign prostate hyperglasia, ischemic kidney failure or
hypertension and kidney failure or hypertension caused by
intoxication, metastasis and growth of mesenchymal tumors,
contrast agent-induced kidney failure, pancreatitis and
gastrointestinal ulcers.
The invention further relates to combinations of endothelin
receptor antagonists of the formula I and inhibitors of the
renin-angiotensin system. Inhibitors of the renin-angiotensin
system are renin inhibitors, angiotensin II antagonists and
angiotensin-converting enzyme (ACE) inhibitors. Combinations of
endothelin receptor antagonists of the formula I and ACE
inhibitors are preferred.
CA 02340167 2001-02-08
0050/49265
The invention further relates to combinations of endothelia
receptor antagonists of the formula I and beta-blockers.
The invention further relates to combinations of endothelia
5 receptor antagonists of the formula I and diuretics.
The invention further relates to combinations of endothelia
receptor antagonists of the formula I and substances which block
the action of VEGF (vascular endothelial growth factor). Such
10 substances are, for example, antibodies directed against VEGF or
specific binding proteins or alternatively low molecular weight
substances which can specifically inhibit VEGF release or
receptor binding.
15 The abovementioned combinations can be administered
simultaneously or sequentially. They can be employed both in a
single pharmaceutical formulation or alternatively in separate
formulations. The administration form can also be different, for
example the endothelia receptor antagonists can be administered
orally and VEGF inhibitors can be administered parenterally.
These combination preparations are especially suitable for the
treatment and prevention of hypertension and its sequelae, and
for the treatment of cardiac insufficiency.
The invention further relates to a structural fragment of the
formula
O R8 R4
6 II I i H
R-C-C-W-C-C-O-~
R' R5 R1
in which the radicals R1, R4, R5, R6, R~, R8 and W have the
abovementioned meaning.
Such structural fragments are suitable as structural constituents
of endothelia receptor antagonists, in particular of mixed
endothelia receptor antagonists.
The invention further relates to endothelia receptor antagonists
consisting of a structural fragment of the formula
0 R$ R4 RZ
II I I H
-C-C-W-C-C-O-~~ Z
R7 Rs Rl X ~ 3
R
CA 02340167 2001-02-08
0050/49265
10
16
in which the radicals R1, R2, R3, R4, R5, R7, Re, W, X, Y and Z
have the abovementioned meanings, covalently linked to a group
which has a molecular weight of at least 40, preferably at least
77.
The good action of the compounds can be shown in the following
experiments:
Receptor binding studies
Cloned human ETA or ETB receptor-expressing CHO cells were
employed for binding studies.
Membrane preparation
The ETA or ETB receptor-expressing CHO cells were proliferated in
DMEM NUT MIX F12 medium (Gibco, No. 21331-020) with 10~ fetal calf
serum (PAA Laboratories GmbH, Linz, No. A15-022), 1 mM glutamine
(Gibco No. 25030-024), 100 U/ml of penicillin and 100 ~g/ml of
streptomycin (Gibco, Sigma No. P-0781). After 48 hours, the cells
were washed with PBS and incubated at 37°C for 5 minutes with
0.05 trypsin-containing PBS. The mixture was then neutralized
with medium and the cells were collected by centrifugation at
300 x g.
For the membrane preparation, the cells were adjusted to a
concentration of 10$ cells/ml of buffer (50 mM tris~HCL [sic]
buffer, pH 7.4) and then disintegrated by means of ultrasound
(Branson Sonifier 250, 40-70 seconds/constant output 20).
Binding tests
For the ETA and ETB receptor binding tests, the membranes were
suspended in incubation buffer (50 mM tris HCl, pH 7.4 with 5 mM
MnCl2, 40 ~.g/ml of bacitracin and 0.2~ BSA) in a concentration of
50 ~.g of protein per test batch and incubated at 25~C with 25 pM
of [125I]-ET1 (ETA receptor test) or 25 pM [125I]-ET3 (ET$
receptor test) in the presence and absence of test substance. The
nonspecific binding was determined with 10-7 M ET1. After 30 min,
the free and the bound radioligand was [sic] separated by
filtration through GF/B glass fiber filters (Whatman, England) on
a Skatron cell sampler (Skatron, Lier, Norway) and the filters
were washed with ice-cold tris HC1 buffer, pH 7.4 containing 0.2~
BSA. The radioactivity collected on the filters was quantified
using a Packard 2200 CA liquid scintillation counter.
CA 02340167 2001-02-08
0050/49265
15
17
Testing of the ET antagonists in vivo:
Male SD rats weighing 250-300 g were anesthetized with
amobarbital, artificially respirated, vagotomized and pithed. The
5 carotid artery and jugular vein were catheterized.
In control animals, the intravenous administration of 1 ~g/kg of
ET1 leads to a distinct blood pressure increase, which lasts for
a relatively long period.
The test compounds were injected i.v. (1 mg/kg) into the test
animals 30 min before ET1 administration. For determination of
the ET-antagonistic properties, the blood pressure changes in the
test animals were compared with those in the control animals.
p.o. testing of the mixed ETA and ETB antagonists:
Male normotonic rats weighing 250-350 g (Sprague Dawley, Janvier)
are orally pretreated with the test substances. 80 minutes later,
the animals are anesthetized with urethane and the carotid artery
is catheterized (for blood pressure measurement) as well as the
jugular vein (administration of big endothelin/endothelin 1).
After a stabilization phase, big endothelin (20 ~g/kg, admin. vol.
0.5 ml/kg) or ET1 (0.3 ~g/kg, admin. vol. 0.5 ml/kg) is
administered intravenously. Blood pressure and heart rate are
recorded continuously for 30 minutes. The clear and long-lasting
blood pressure changes are calculated as the area under the curve
(AUC). For determination of the antagonistic action of the test
substances, the AUC of the substance-treated animals is compared
with the AUC of the control animals.
The compounds according to the invention can be administered
orally or parenterally (subcutaneously, intravenously,
intramuscularly, intraperitoneally) in a customary manner.
Administration can also be carried out through the nasopharynx
using vapors or sprays.
The dose depends on the age, condition and weight of the patient
and on the type of administration. As a rule, the daily dose of
active compound is between approximately 0.5 and 50 mg/kg of body
weight in the case of oral administration and between
approximately 0.1 and 10 mg/kg of body weight in the case of
parenteral administration.
CA 02340167 2001-02-08
0050/49265
18
The novel compounds can be used as solids or liquids in the
customary pharmaceutical administration forms, e.g. as tablets,
film-coated tablets, capsules, powders, granules, sugar-coated
tablets, suppositories, solutions, ointments, creams or sprays.
These are prepared in a customary manner. In this case, the
active compounds can be processed using the customary
pharmaceutical auxiliaries such as tablet binders, fillers,
preservatives, tablet disintegrants, flow-regulating agents,
plasticizers, wetting agents, dispersants, emulsifiers, solvents,
release-delaying agents, antioxidants and/or propellants (cf. H.
Sucker et al.: Pharmazeutische Technologie [Pharmaceutical
Technology], Thieme-Verlag, Stuttgart, 1991). The administration
forms thus obtained normally contain the active compound in an
amount from 0.1 to 90~ by weight.
Synthesis examples
Example. 1
Methyl 2-hydroxy-3,3-diphenyl-3-(2-phenyl-[1,3]-dioxolan-2-yl-
methoxy)propionate
p-Toluenesulfonic acid (0.50 g, 0.27 mmol) was added with
ice-cooling to a solution of 2-phenyl-[1,3]-dioxolan-2-ylmethanol
(1.98 g, 11.0 mmol) and methyl 3,3-diphenyl-2,3-epoxypropionate
(4.71 g, 13.2 mmol; purity according to HPLC: 71g) in anhydrous
dichloromethane (100 ml) and the mixture was stirred at 0°C for 15
minutes. The resulting solution was washed with sodium hydrogen-
carbonate solution, and the organic phase was separated off and
dried over magnesium sulfate. After filtering off the drying
agent, the solvent was removed by distillation; the residual
crude oil (4.70 g) was reacted further without further
purification.
Example 2
2-Hydroxy-3,3-diphenyl-3-(2-phenyl-[1,3]-dioxolan-2-ylmethoxy)-
propionic acid
Methyl 2-hydroxy-3-(2-phenyl)-[1,3]-dioxolan-2-yl-methoxy)-
3,3-diphenyl propionate (4.60 g, crude) was dissolved in
dioxane/water 2:1 (45 ml) and treated with sodium hydroxide
(300 mg, 7.50 mmol). The mixture was warmed to 40°C and stirred
for one hour. For work-up, it was diluted with water (150 ml) and
extracted twice with ethyl acetate. The aqueous phase was
acidified with citric acid and extracted twice with ethyl
acetate. The extracts obtained from the acidification were dried
CA 02340167 2001-02-08
0050/49265
19
over magnesium sulfate and the solvent was removed by
distillation. 4.00 g of a crude oil were obtained, which was
reacted further without further purification.
Example 3
2-(4-Methoxy-6-methylpyrimidin-2-yloxy)-3,3-diphenyl-3-(2-phenyl-
[1,3)-dioxolan-2-ylmethoxy)propionic acid
50~ strength sodium hydride (240 mg, 5.00 mmol) was added in 3
portions in the course of 3 minutes to a solution of 2-hydroxy-
3-(2-phenyl-[1,3]-dioxolan-2-ylmethoxy)-3,3-diphenylpropionic
acid (1.00 g, 1.62 mmol at 68~ purity according to HPLC) in
anhydrous DMF (15 ml). The mixture was stirred for 5 minutes and
2-methanesulfonyl-4,6-dimethylpyrimidine (421 mg, 2.00 mmol) were
then added in portions. The mixture was stirred at room
temperature for 16 hours. For work-up, the contents of the flask
were poured onto ice water, then acidified with citric acid and
extracted twice with ether. The organic extracts were dried over
magnesium sulfate and the solvent was removed by distillation.
1.75 g of an oil remained, which was further purified by flash
chromatography and subsequent crystallization from
ether/n-hexane. The title compound was obtained as a colorless
solid (750 mg, 85~ yield).
1H-NMR (200 MHz, CDC13): 7.5 - 7.7 ppm (2 H, m), 7.2 - 7.4 (13 H,
m), 6.3 (1 H, s), 6.2 (1 H, s), 4.2 - 4.4 (2 H, m), 4.1 (1 H, d),
3.9 (3 H, s), 3.8 - 4.0 (2 H, m), 3.6 (1 H, d), 2.4 (3 H,s).
Example 4
2-(4-Methoxy-6-methylpyrimidin-2-yloxy)-3-(2-oxo-2-phenylethoxy)-
3,3-diphenylpropionic acid
p-Toluenesulfonic acid (50 mg) was added to a solution of
2-(4-methoxy-6-methylpyrimidin-2-yloxy)-3-(2-phenyl-[1,3]-
dioxolan-2-ylmethoxy)-3,3-diphenylpropionic acid (600 mg,
1.11 mmol) in dioxane/water 1:1 (20 ml) and the resulting mixture
was stirred at 80°C for 2 hours. After cooling, the mixture was
diluted with water and it was extracted twice with ether. The
combined organic phases were dried over magnesium sulfate and the
solvent was removed by distillation. The residue (550 mg) which
remained was purified by crystallization from ether/n-hexane,
subsequent flash chromatography and crystallization from
ether/n-hexane again. The title compound was obtained as a
crystalline solid (163 mg, 30~ yield).
CA 02340167 2001-02-08
0050/49265
20
20
1H-NMR (200 MHz, CDC13): 7.2 - 7.9 ppm (15 H, m), 6.2 (2 H, s br),
5.1 (2 H, m), 3.7 (3 H, s), 2.2 (3 H, s).
ESI-MS . M+ = 498
The following compounds were prepared analogously:
Example 5
10 2-(4,6-Dimethylpyrimidin-2-yloxy)-3-(2-oxo-2-phenylethoxy)-3,3-
diphenylpropionic acid
1H-NMR (200 MHz, CDC13): 7.8 ppm (2 H, d), 7.2 - 7.7 (13 H, m),
6.7 (1 H, s), 6.3 (1 H, s), 5.2 (1 H, d), 4.9 (1 H, d), 2.3 (6 H,
15 s).
ESI-MS: M+ = 482
Example 6
3-[2-(4-Bromophenyl)-2-oxoethoxy]-2-(4,6-dimethylpyrimidin-
2-yloxy)-3,3-diphenylpropionic acid*
1H-NMR (200 MHz, CDC13): 7.7 ppm (2 H, d), 7.6 (2 H, d), 7.2 - 7.5
25 (10 H, m), 6.7 (1 H, s), 6.2 (1 H, s), 5.1 (1 H, d), 4.9 (1 H,
d), 2.3 (6 H, s).
ESI-MS: M+ = 560
30 Example 7
3-[2-(4-Bromophenyl)-2-oxoethoxy]-2-(4-methoxy-6-methylpyrimidin-
2-yloxy)-3,3-diphenylpropionic acid*
35 1H-NMR (200 MHz, CDC13): 7.7 ppm (2 H, d), 7.6 (2 H, d), 7.2 - 7.5
(10 H, m), 6.2 (1 H, s), 6.0 (1 H, s), 5.2 (1 H, d), 4.9 (1 H,
d), 3.7 (3 H, s), 2.2 (3 H, s).
ESI-MS: M+ = 576
* In the synthesis of 4-bromophenyl-substituted derivatives,
boron trifluoride etherate was used instead of p-toluenesulfonic
acid for the final acetal cleavage.
CA 02340167 2001-02-08
. 0050/49265
21
Example 8
Benzyl 2-hydroxy-3-[(methoxymethylcarbamoyl)methoxy]-
3,3-diphenylpropionate
Boron trifluoride etherate (0.10 ml) was slowly added to a
solution, cooled to -78°C, of 2-hydroxy-N-methoxy-N-methyl-
acetamide (1.19 g, 10.0 mmol) and benzyl 3,3-Biphenyl-2,3-epoxy-
propionate (3.88 g, 11.0 mmol; purity according to HPLC: 94~) in
anhydrous dichloromethane (100 ml). The mixture was stirred for
two hours and in this time gradually warmed to -20°C, and the
reaction was stopped by cautious addition of aqueous sodium
hydrogencarbonate solution. The organic phase was washed with
sodium hydrogencarbonate solution and dried over magnesium
sulfate. After filtering off the drying agent, the solvent was
removed by distillation; the residual crude oil (5.50 g) was
reacted further without further purification.
Example 9
Benzyl 3-[(methoxymethylcarbamoyl)methoxy]-2-(4-methoxy-6-methyl-
pyrimidin-2-yloxy)-3,3-diphenylpropionate
A solution of benzyl 2-hydroxy-3-[(methoxymethylcarbamoyl)-
methoxy]-3,3-diphenylpropionate (1.35 g, crude) was treated with
ice-cooling with potassium carbonate (365 mg, 2.64 mmol) and,
after 10 minutes, with 2-methanesulfonyl-4-methoxy-6-methyl-
pyrimidine (320 mg, 1.45 mmol). Subsequently, it was stirred at
0°C for 30 minutes and then at room temperature for 16 hours. The
mixture was diluted with water, acidified with citric acid and
extracted twice with ether. The combined organic phases was [sic]
backwashed with water and dried over magnesium sulfate. After
distilling off the solvent, a foam (1.60 g) remained, which was
purified by flash chromatography and subsequent crystallization
from ether/n-hexane; 650 mg of the title compound were obtained.
Example 10
Benzyl 3-[2-(3,4-dimethoxyphenyl)-2-oxoethoxy]-2-(4-methoxy-
6-methylpyrimidin-2-yloxy)-3,3-diphenylpropionate
A 1-molar solution of 3,4-dimethoxyphenylmagnesium bromide in
tetrahydrofuran (0.60 ml) was added at room temperature to a
solution of benzyl 3-[(methoxymethylcarbamoyl)-methoxy]-
2-(4-methoxy-6-methylpyrimidin-2-yloxy)-3,3-diphenylpropionate
(250 mg, 0.38 mmol, purity according to HPLC: 86~) in anhydrous
tetrahydrofuran (25 ml). After stirring for 10 minutes, only
CA 02340167 2001-02-08
0050/49265
15
25
22
partial conversion was observed, therefore a 1-molar solution of
3,4-dimethoxyphenylmagnesium bromide in tetrahydrofuran (0.60 ml)
was added dropwise again. The mixture was stirred for a further
10 minutes, then the solvent was evaporated. The residue was
5 taken up in ethyl acetate/ether 1:2. After filtering off
undissolved matter, the solvent was removed by distillation, and
the oily residue (400 mg) was purified by flash chromatography.
The title compound was obtained as a foam (125 mg, 49~ yield with
95~ purity according to HPLC).
1H-NMR (400 MHz, CDC13): 7.5 - 7.7 ppm (4 H, m), 7.4 (2 H, d), 7.2
- 7.3 (9 H, m), 6.9 (2 H, d), 6.8 (1 H, d), 6.3 (1 H, s), 6.2 (1
H, s), 5.4 (1 H, d), 5.0 (2 H, m), 4.7 (1 H, d), 3.9 (6 H, s),
3.7 (3 H, s), 2.3 (3 H, s).
The compounds in Table I can be prepared analogously or as
described in the general section.
Example 11
Receptor binding data for the compounds listed below were
measured as in the binding test described above.
The results are shown in Table 2.
Table 2
Receptor binding data (Ki values)
Compound ETA [nM] ETB [nM)
Example 4 10 114
Example 5 31 119
Example 6 18 168
Example 7 13 106
I-10 1.7 20
I-114 1.3 21
45
CA 02340167 2001-02-08
0050/49265
23
O v~c~v~O O O O O O O v~O O O
~ z z z z z z z z z z z z z z z
~tz z z z z z z z z z z z z z z
N U U
' x'
N
U U U U U U U U V
N U V (j U U U N
N
x ~
x
x
Cj
N T'
~
v ~ o~~ a~a~a~U a~s a~~ya~U
CG~ O ~ O ~ ~ ~ ~ w ~ U
x x
a~~~a~~ ~~ a~asa>>,.,a~ v
O O ~ O O
C~~ O U O O U ~ O W U ~
N M
N ~:
y, ~ ~ x x x x x ~ x x x z x x x x
x
Y T
O
c~~ x x ~ x oa~ x x x x x x x x
x~-x
0:- U -f~:
0: - U C4 ~ ~ ''
s a
c C C C ~ C '
T
-C~ ~,.Ct .GN ~ ~ ,~S ~ C C C
CL~ s n.a ~ ~ ,=n.~ C.~ a>a~v
' o c n
. . .
~ ~
O ' Z O O O U ~ O ' O U U U
' '
'o~ '~I I I I ~ I I ~:I '~~ I I
'
~'M N M M M ~tM M M ~YM N M N
T
s
G
_
'~ T T
C C
C v
7, ~ t
T ? ~ T T ~
T ?,~,T T T ~ T T C , I C C _
C C C C C C c C C ~ C Gi,v ~ U
a~a~v ~ a~a~ ~ v a~
C;tea ~ = L a Z a n~o cuet oLa t
a . .,a a . ... ..~ .,. ~
, o . ..
x x x x x x Z x x x x x x x x
O O O O O O ~ O O O O O O O O
O O V V O 0 0 0 0 0 0 0 0 0
U U U U
"G7 '"~ M
0 -.N M ~r~n~ r.x o~
H z " ' . . . ' . . ' . " . .
CA 02340167 2001-02-08
0050/49265
24
cnO O O O O O cncnO O O O O O cnO O O O cnO O O O cn
~' z z z z z z z z z z z z z z z z z z z z z z z z z z
~t z z z z z z z z z z z z z z z z z z z z z z z z z z
U U N U
x x x x U ~ = x x ~ = x x x x x x U = x x x T x x x
N U U U U ~ U U U U U U U U U U U U U U U U U
, U U I
Z' i', ~I
x x U x
N r.~~ Q T Q 'y ~ x O T T T
U U
C~ O ~ ~ ~ O ci~~ ~ U ~ O ~ O ~ t.~w ~ U w ~ O
x x
N ~ j~ , N N N ~ N O N N T T ~ T N
r" O O
O O U O O x O w ~ U O O ~ O U O O w w V O w ~ O
c~ x x x x ~ x z x x x x x x x x x x x ~ s z x x x x x
i~ x x x x ~ x ~ x x x x x x x x x x x ~ x x x x x x x
T
T N C C
C C 41 >'
T ~' T S N'C C _ = T
C C C
a'S s ~ s ~ C >'T ~'C C C'N s
v v y
a a a v o.n.~ c a = ~ ~ v s ~ ! s o
I .,~ I ~ I I ~ , ~ T ~ 0.a ~ a, ~ . ~ ~,.cv >
I
_ _ , ,
U U U O ~;~; ~;L W ~ ~ ~
'
s s ~ a.T ;r I >, ; ~ v i v v
I I , I v v ~ y ~ p T v c L~
a a ~y ~ i1 I
I A O I D A ~ ~ ' ~ I a ~ C = ~ ~ s ~ I CDo o D ~ c>
~
" , , :.
U I I U 1 I 1 I ~ I ( ! Q O O U ' I ~
U
M d' ~h~ ~ ~ d~'d~, . W G) Pr5~ ~ T T V
L t
I ~1M ~ M I I I 1 I O I 1 O , M U U M ~ 'd'
M ~ M ~ M M ~t d'
~ f ~ M M M M M .. .
T
C
d T
_
T _
L
I t T
T
C
C T V T y T C .~C ~,d'
y C ~'.C t C ~ Q. ~ "
y _ 0._y_ _ _ ~ ~ ~'L _ _ _ L _ _ N .>_
d C T d ~ >
T T T I O.T 7,T T . ~ ..T T >,~ T T 7,~ T L ,
1 C 1 C C
C C C _ 1 C C C C 1 ~ G>:.C C C ~ C C C O C v ts.c-v v
v v v U tiv v v v t_ a~v v v v v v
t L t 1 . .J:.JrL t , I 1 -CL t ~ t L L I L .C1 '~.Ct
~ I 1 z 0.0 z 0 Ch
, G,0.O..~ 'V'0..GL0.C.~t~t~tG1.O.,0. 0ØCLM ..~ ..
v v
x x x x z x x x x x ~ x x x x x ~ z x x x x x x x x
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0
x a o N M ~rv m:r-x a o -~N M ~ I w.~ x a o -
O -'~ f'IN N N ('JN C'lN C'I('IM M M M M M M M M M V'
~ ~
I I 1 , 1 ~ 1 ~ I I 1 , , ~ I ~ I I I , , 1 I I
CA 02340167 2001-02-08
0050/49265
O O O O O O O O O O O O O O O O O O O v~O O
O O O cn
~ z z z z z z z z z z z z z z z z z z z z z z z z z z
>tz z z z z z z z z z z z z z z z z z z z z z z z z z
V U U V U U
U V ~ N "l
N I 1 '7 ~ I I
' '
x x x ~ _ x x x s x x x ~ x x x ~ x x x x ~ x x ~
U V V U U V U U V U U U U U U U U
N U U U I U (~ N ~ N
l 1 '
x N c
x n1 N x x .,., 1
x
x z
N N N U V
U U V
N 1 I
O O
~ n ~ V a~ v ~ U v a~~ V a~v v a~U s
T ,~
c
~ . ~ U U ~ O ~ ~ O ~ ~ ~ ~ tit w
w
x x
ru a~a~ a~a~ al a~a~a~ a~a~a~~ a~~,a~>,~ >,
O
~ x ~ ~ s ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ x ~ t ~ _ ~ t ~
x w W w O O O ~ O O O ~ O O O U O W O w O w O
U O U O O U
ixx x x x x x x x x x x x x x x x x x x x x x x x x x
w x x z x x ~ x x x ~ x x x x x x x x
r~x x x x x x x
T
C
v
t
G
T I
t T _ C N _
C- ~ T t x T T
t Q C C
_ T - c U c
T O C ~ L C N 1 .C T T .C N .C
_ S ?,~ ~ ..~x n y y T G- L C.
G a,c ~ a -cw ~ U r I .>_ .>= - y a p _a~
~ v
. ~ ' ~ 1 p , ~ > fl' a ~ > c ,
I
v . T U , I , a
o
0 0 o v z o ~ ~, ~ w ~ ~ I o I o
s ~
y a~
O ,T.~ ~ N O ~ a ~ ~ O o ~ ~ ' Ca' ~ CD
~ ~
~ a a F y ~ ~ ~ ~ ~ z ~ Z a, U ~.~ U O Z ~ Z O
,o . . ~ v . L ~ V
CGN N d'M N M M d'CV M M d'M ~ '~t0..V'M V ~ N ~ M N ' M
T
C
T
t
O_ _ _ d
y T
T
T
C
I G
~ G
C ~ '~ >'_ >'T T
C
T C T T C C
d C C ~y X X C t C C t y
I ~ ~ I v v ~ d.~ = O'O d
U T fIT _ _ T T N. Q T T T T p ~ _ Qjd 0-N _ T I T T
T T C- I
C I G C C G C 1 C C C C I I I _ C C C
~ V Lz~ ttLt~ U v U v v
~r y GLv v v a~a~L, ,~.v v v v T T . I , ..I I s I .c
c s t s e s s I I i
~ I O I t .c.ØG I M O . C 0.U U calM d'C ~'M G.d 0Ø
CSO N O
r cf. ~ 0.. ... . , . .
v v
x x x x x x x - x x x x ~ x x x ~ x x x x x x x x x
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
~ 0 0 0 0 0 0
rlM ctV1'~.(~OGQ'"''~ C'lM ~tV I~00.r"1C ~ rlM ~YI!1~.Ot'
o ~ ~r~ v v ~rv d-~, ~nv,~,~nInIrInInv : s ~ .~..ow.~
I
~ I ~ I 1 I I I I 1 I I I I I ~ I , I I , I ~ i I
CA 02340167 2001-02-08
0050/49265
26
O O cnO O O O ~nO vm~ O O w O O O O O O O v~O O O
~ z z z z z z z z z z z z z z z z z z z z z z z z z
v
x z z z z z z z z z z z v v z z z z z z z z z z z
U V U
''' '..' I U
I
' ~ ~ U
4 '
U x Z i i x U x ~ x x x x x i U ~ x x x
N U U U U U U I U U U Z z z U U U Z U U , U U U U U
N
N I
I
N
U U U U
n! I nlI
~ O
U O U
fxU ~ ~ ~ O ~ O O W O ~ ~ O ~ ~ ~ ~ ~
x
T T T
~ y y ~ ~ ~ ~ ~ y ~ ~ ~ x -cy x
N y x ~ ~ 0 0 o w o ~ Li.x o o w o x x w ~ U o
c ~ U
c o x o x
ccx x x x x x x x x x x x z x x x x x x x x x x x x
~ x x x x x x ~ x x x x x x x x x x x x x x x x x x
T
C c
G C C C C C C C L L
c j,~~ _ >, T y _
S t ~ y y ~ ~' a c c
y y y y . . ' c -~ ~' L ~,
d Q C c y ~
O
d d G fl. ~- . . Q. ~ ~ a y G p,
T T T 7
U U U U V U U U ~ ~ _ U y.~ ' ryX U o.
Q. C ,~_I_~_I_ y y y y o ! >'y VrT y C I S S N
d
1 I I Q G7O D ' ~ ~ ~ ~ a.O U ~ U L
U U U I I I I ~ I I I I ~ I Q i Q W o 0.y ~ ' o
M 'C - ' ~ V V ' !1~ !t ~ U ~
?
1 1 I ~ ~ _.~ h ~ ~ I I I I 1 I S , I
CZiN M V N M M M GLM M M M cYM M M ~ M M ~ OrM U M 'cr
7, T
C C
y y
L L
I
, I U
u.
a tr >, _ _ '
_ _ _ T T
'
"
~ T C -c T T y y 7,
- - c
~ c y ~ c c s _.
~ s s s .
_ _ _ _ _ _ _ _ _ _ _ _ _ _
T T T T T '~n a ?a~ T C-p-T T ~ T T T T T T T q~G.
~ v v v ~ fsL:_ v W y u czv v ~ v v v v v ~ v ~ Li.
U
.cs L L r . , I L I ~ . , s L I L s s L s s L ! I
~ I ! I I O 0.G 0 G C G ~
r C.C. LLO.Ø.M ct'~tCL~ 0..~ ~ G..Q,N .. . . . . . 1 ch
t
y y
x N x x x z x x x x x x x x x x x x ~ x x x x x x
o ~ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
--,
O .~ 0 O O O O O O O O O O O O O O O V O O O O O O
~
0oa c --~N M m n ~ r ooa ~ m cn v !w: r oca ~ --~c~~
p ~ ~ r r r r r r r r r r x x o000 00occcr o000~ a a
I I ! I I I I , I ~ ~ I I , I ! I I I ! ~ I ~ ! I
CA 02340167 2001-02-08
0050/49265
27
3 0 0 ~ 0 0 0 ~ 0 0 0 0 0 0 0 0 0 0 ~ 0 0 0 0 0 0 0
~ z z z z z z z z z z z z z z z z z z z z z z z z z
~ez v z z z z z z z z z z v z z z z z z z z z z z z
V U U U ~;~, 'r' U U
~ 1 t'I1 x x x N I w
x x x U "4'x U = x x ~i~ix U .~x ""U '",,"".rl~ix
N Z Z U U U I U U 1 U U U Z U U U U U U U 1 U U U U
N f~l N
I 1 1
rl x,rl x N
U U U U U
U
v x O > ZIO > v ~~ O >
, , ,
r~v ao~ v v U L U v ~yv v v .~v ~ v v U v L v
C~G~ ~ O ~ ~ W ~ U ~ ~ ~ W ~ZO ~ ~ ~ tip
~ ~ ~ v v ~ ~ ~ ~ ~ ~ v ~ s v ~yv v ~ a~v
N o 0 0 ~ x ~ x ~ o o ~ o o ~ o w x x ~ U ~ ~ o ~ ~
x
v
~ x x x x x x x x x ~ x x x x x x x x ~ ~ x x x x x
n v s v v
x x x x x x x x x x ~ x x w x x x x x ~ ~ x x x x x
a
a
v v
I > > ~ c c V
x
X >, G , T , . _ r i t L C
X C
v - ~ . 1 ~l>, a a
o.
a o v c v ~ n.n a.ai~ O U ~ _ _ aiv >,' >,o-
~ T ~ ~
, V T ~ = T T . U I i1T T C C C
I U U
v >,>,~ a .cG,T O O O ~ ~ s O ~ s O s vcU
O
a.I ; :o;~c v ~ o O 1 x zl u..L .cI ~,c I a . !.
c
~ , ~ ~ ; ~ ~ a.~ v CO1 v O U :~cps'GlL I CaI I O
v Gr. _
~ U , ,,, ~ I ~ Q ~ 1 ~ ~ ~ o z z I w a I v a I
~.c.~-
~'I I 1 I 1 1 ~ 1 I 1 ~ I I 1 I I I ~ 1 I ~ 1 1 M
~
U 'Ctct N ~tM N M d'M M M 'd'M V'~ ~ N M M C'IM M V'N
T
C
v
r
G _
C 1
C C T
> > :' s ~ ~ v
, , . ; c c .
~ s y ~ s .vc ~ a
InG.T T T G _ T _ 7,~ _ _ _ 7,_ ~ _ _ T IGIGT 7,
T T ~ T T T T T 7,
I C C C 1 C C C C C C C C C C C C _ _ C C
~ Gr.v v v tr.v v v v cnv v v v v O v v v U U v v O U
,sI .CL S I S -CS S I .C-C~ S ~ I L -CL 1 I .C~ I I
~tfY0. C.d'O.C1Ø0.V'0,C1,C.OØ.M O.GYG ~tctGØ.M ct
v
x x N x x .~x -~~~x x x x x x x x x x x x z N x x
O O ~ O O O O O O O O O O O O O O O O O O O L O O
~-.
O O ~ O O O O O O O O O O O O O O O O O O O ~ O O
.~
~~~t~w:,t~x a _~. ~nM rr.n',n
M ~ ~w :,r ~ a o 0 ' 0 o c 0 0 0 0 - ,..~.
_
_ _ _ _ _ _ _ _
C a a c~ a a a o~-~,-a.-.~-. --,.-.-~-.
.
I 1 1 1 I I I I I I 1 I I I I I I I 1 1 1 I I 1 I
CA 02340167 2001-02-08
0050/49265
28
3 0 0 ~ 0 0 0 0 0 0 0 0 0 0 0 0
~ z z z z z z z z z z z z z z z
~ez v v z z z z z z z z z z z z
", U
1
U
1
'
~ix x V x w'x x ~ ~ x
N U Z Z U U U I U U U U U U Z U
x m
U U m
I .-.
ty I M
x cn
U
U
x ~ O x x U ~ ~ ~ ~ ~ Z x
N
M
v a~ v ~ > > ~ a~
. a~a~, V
o s s o
c~z ~ o o x o x ~ w ~ ~ w z
c~x x x x x x x x ~ x x x x x x
~ x x x x ~cx x x ~ x ~ x x x ~
T T
_ T 7,y j,
T T T
G C C .CC _
c c v y a~n a~ T a >, >,
. c a
v ' -c-ct I .c>.c T ,
d d
G C a y ~-~ ~ ~ a~~ d d
I 1
L ~ G p~ I 1
U U U U U v a o. I c
_ I T
I I v v a ~' '
D _ v ~ ~ v C ~ ~ w i,y c ~ C~
D ~ ~ ci~ U ~ ~ ~
O
O O O
M M M M M d' M M M ~ O M
T
C
T S ~ C N C
a T _ _ a~ " >, v _
I c ~
T T d C C d. T
a~
v C
S s = I j~~ ~ C I T 7,v
G C ~ d ~
-''c.n.V ~?, ' ~ ; ~ v c.
I
D ~,s aJaiQ ~ ~ ~ ~ _~Q o o v
u,W ~ ~ I v u.v7O U
~ ~ ~ T T
I I I I ~ I I I I 1
CLM M V'f'!N M 0.d'Wit'M V'M U U M
x x x x x x x x x z x x x x x
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
o o 0 0 0 0 0 0 0
~ ~ 0 0 0 0 0
x a o --~N M ~ v;..~.r-x a o - m
fVfVfVN N (V(VN_C_'JN_M_ M M
p ,_.a~. .-~_. .r~.
1 I I 1 1 I 1 I I I 1 I 1 1 1
